Live dead cell imaging kit 488 570

SMP samples were submerged in growth media and were equilibrated to 37 °C for 24 h prior to cell seeding. HUVECs were then plated on 1cm diameter SMP substrates in 24-well plates and were allowed to attach. Cells were seeded at a seeding density of 1 × 10⁵ cells/mL per well. Daily monitoring of cell-adherent SMPs using transmission microscopy allowed for the qualitative assessment of proper cell growth and the absence of contamination. Cell viability was quantitatively assessed at two time points, approximately 24 h after plating and again at approximately 72 h. Complete cell medium was changed daily to ensure that cells received consistent nourishment during the study. The Live/Dead Cell Imaging Kit (488/570) (Life Technologies, Carlsbad, CA, USA) was used to assess endothelial cell attachment and viability through fluorescent staining. Live cells, which were actively attached to the substrate, emit green fluorescence, while dead cells fluoresce red. Images were obtained using an EVOS FL Cell Imaging System (Life Technologies, Carlsbad, CA, USA). At least three images from three replicate experiments were used for cell attachment counting using ImageJ software (NIH).

Cells were seeded in a 24-well plate overnight. After treatment, cells were stained with the LIVE/DEAD Cell Imaging kit (488/570) (Thermo Fisher Scientific, Inc., Waltham, MA) as described previously [49 (link)]. After incubation at room temperature in the dark, the cells were observed under EVOS FLoid Cell Imaging System (Thermo Fisher Scientific) and measured for live cells (green) with Green-Light Channel and dead cells (red) with Red-Light Channel.

Human packed red blood cells were donated by the Faculty of Medicine Blood bank, at Prince of Songkla University, Thailand. Carboxy-terminated 50:50 poly(d, l-lactide-co-glycolide) (PLGA) polymer 0.66 dL/g was purchased from LACTEL Absorbable Polymers (Birmingham, AL, USA). Doxorubicin hydrochloride was purchased from Pfizer Laboratories (New York, NY, USA). Phosphate buffer solution (PBS) was acquired from Thermo-Fisher (Waltham, MA, USA). Both the bicinchoninic acid (BCA) assay kit and paraformaldehyde were procured from Millipore Sigma (St. Louis, MO, USA). Streptavidin, Streptavidin-Fluorescein Isothiocyanate (Streptavidin-FITC), biotinylated anti-EpCAM monoclonal antibody was bought from Biolegend (San Diego, CA, USA). The MTS Assay Kit (Cell Proliferation) (colorimetric) was purchased from Abcam (Cambridge, MA, USA). MCF7 breast cancer cells and PCS-201-010 dermal fibroblast were purchased from ATCC (Manassas, VA, USA). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotin(polyethylene glycol)-2000] (ammonium salt) (DSPE–PEG-biotin) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). LIVE/DEAD Cell Imaging Kit (488/570) was bought from Thermo-Fisher Scientific (Waltham, MA, USA). CellTiter-Glo^® was purchased from Promega (Madison, WI, USA). Other chemicals and reagents were from Millipore Sigma (St. Louis, MO, USA), or Thermo-Fisher (Waltham, MA, USA).